Synergistic immunosuppressant composition containing a 2,2&#39;-bi-1H-pyrrole compound

ABSTRACT

A product containing: (a) an immunosuppressant agent (A) and (b) at least one immunosuppressant 2,2′-bi-1H-pyrrole compound (B) having formula (I) wherein R 1  is hydrogen, phenyl, C 1 -C 20  alkyl or C 2 -C 20  alkenyl, wherein the alkyl and alkenyl groups are unsubstituted or substituted by 1 to 3 substituents, which are the same or different, chosen independently from halogen, C 1 -C 6  alkoxy, hydroxy, aryl and aryloxy; R 2  is hydrogen, C 1 -C 6  alkyl, cyano, carboxy or (C 1 -C 6  alkoxy)carbonyl; R 3  is halogen, hydroxy or C 1 -C 11  alkoxy unsubstitued or substituted by phenyl; R 4  is hydrogen, C 1 -C 6  alkyl or phenyl; each of R 5  and R 6 , which are the same or different, is independently hydrogen, C 2 -C 20  alkanoyl, C 3 -C 20  alkenoyl, phenyl, C 1 -C 20  alkyl or C 2 -C 20  alkenyl; or two of R 4 , R 5  and R 6 , taken together, form a C 4 -C 12  polymethylene chain, which is unsubstituted or substituted by a C 1 -C 12  alkyl, by a C 2 -C 12  alkenyl or by a C 1 -C 12  alkylidene group; or a pharmaceutically acceptable salt thereof; as a combined preparation for simultaneous, separate or sequential use in immunosuppressant therapy, said preparation having a potentiated immunosuppressive activity with respect to products containing either the immunosuppressive agent (A) or the 2,2′-bi-1H-pyrrole immunosuppressive compound (B).

This application is a 371 of PCT/EP97/04884 filed on Sep. 2, 1997.

The present invention relates to a combination preparation containing:

(a) an immunosuppressant agent (A), and

(b) an immunosuppressant 2,2′-bi-1H-pyrrole compound (B), as hereindefined.

The preparation has an increased immunosuppressive activity, relative tothe sum of the effects produced by immunosuppressant drugs (A) or (B)used alone, allowing greater immunosuppressive activity with reducedtoxicity.

BACKGROUND OF THE INVENTION

Presently, the most commonly used agents for preventing and treatingrejection phenomena associated with organ and tissue transplantations,graft-versus-host diseases and autoimmune diseases are immunosuppressivedrugs, e.g. cyclosporin A (CsA), FK506, azathioprine (AZ), methotrexate(Mtx), rapamycin (R), mycophenolate mofetil (Mac) andglucocorticosteroids (Gluc).

All these drug therapies are limited in effectiveness, in part becausethe doses needed for effective treatments may increase the patient'ssusceptibility to infection by a variety of opportunistic invaders and,mainly, because of the side effects caused by its direct toxicity. Forinstance, despite various successful results, a serious limitation tothe wider application of CsA in these indications is the toxicity ofthis substance. In the first place, its marked nephrotoxicity which insome cases is irreversible has to be mentioned here, but also otherphenomena such as hypertension, nausea, diabetes, diarrhoea, tremor,tingling or gingival hypertrophy (Palestine A.R. et al.: Am.J.Med. 77(1984), 652-656), and lymphomagenesis represent complications to betaken seriously, which usually cannot be avoided even with systematicchecking of the serum level. In addition, opportunistic infections haveto be considered (Dawson T. et al.: J. Rheumatol. 19 (1992), 997), sothat by critical benefit-risk assessment an otherwise advantageous CsAmedication in many cases has to be sacrificed. FK506 (Tacrolimus) is amacrolide which exerts largely similar effects as CsA, both with regardto its molecular mode of action and its clinical efficacy (Liu J.:Immunol. Today 14 (1993), 290-295; Schreiber S.L. et al.: Immunol. Today13 (1992), 136-142); these effects, however, may be found already atdoses which are less by the factor 20 to 100 compared to CsA (PetersD.H. et al.: Drugs 46 (1993), 746-794). The same is true for rapamycin(R) which again is a macrolide binding intracellularly to the sameimmunophilin as FK506, although the following biochemical events arediffering somewhat (Morris R.E.: Transplant. Rev. 6 (1992), 39-87).

Accordingly, it would be desirable to have a drug capable ofpotentiating the action of currently used immunosuppressive agents.Ideally, such a drug would increase the efficacy of suchimmunosuppressive agents and also decrease deleterious side-effects byallowing administration of lower dosage levels.

After an extensive study on the possibility that the effect of animmunosuppressive agent (A) in the present invention is improved bycombining it with a variety of compounds, the present inventor hassurprisingly discovered that the effect of an immunosuppressive agent(A) is significantly improved and side-effects can be decreased byco-administering it with at least one 2,2′-bi-1H-pyrrole compound (B),as herein defined.

2,2′-Bi-1H-pyrrole compounds (B), according to the present invention areimmunosuppressive agents which are known, e.g., from WO 95/17381. Suchdescription also shows a combined use of an immunosuppressant agent (A)and a 2,2′-bi-1H-pyrrole compound (B), in immunosuppressive therapy.However, WO 95/17381 neither shows, nor suggests, that said combined usecause synergistic increase in effect or decrease side-effects inimmunosuppressive therapy. In particular, WO 95/17381 neither shows, norsuggests, that the same therapeutic effect obtainable by the combineduse of therapeutically effective amounts of an immunosuppressant agent(A) and a 2,2′-bi-1H-pyrrole compound (B) can be similarly also obtainedby co-administration of doses by itself inactive of the same twoimmunosuppressant agents (A) and (B).

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides a product containing:(a) an immunosuppressant agent (A) and (b) at least oneimmunosuppressant 2,2′-bi-1H-pyrrole compound (B) having the followingformula (I)

wherein

R₁ represents hydrogen, phenyl, C₁-C₂₀ alkyl or C₂-C₂₀ alkenyl, whereinthe alkyl and alkenyl groups may be unsubstituted or substituted by 1 to3 substituents chosen independently from halogen, C₁-C₆ alkoxy, hydroxy,aryl and aryloxy;

R₂ represents hydrogen, C₁-C₆ alkyl, cyano, carboxy or (C₁-C₆alkoxy)carbonyl;

R₃ represents halogen, hydroxy or C₁-C₁₁ alkoxy unsubstituted orsubstituted by phenyl;

R₄ represent hydrogen, C₁-C₆ alkyl or phenyl; each of R₅ and R₆independently represents hydrogen, C₂-C₂₀ alkanoyl, C₃-C₂₀ alkenoyl,phenyl, C₁-C₂₀ alkyl or C₂-C₂₀ alkenyl, wherein the alkanoyl, alkenoyl,alkyl and the alkenyl groups may be unsubstituted or substituted by 1 to3 substituents chosen independently from halogen, C₁-C₆ alkoxy, hydroxy,aryl, aryloxy, cyano, carboxy, (C₁-C₆ alkoxy)carbonyl, (C₃-C₄alkenyl)carbamoyl, aralkylcarbamoyl, arylcarbamoyl and —CONR_(c)R_(d) inwhich each of R_(c) and R_(d) independently is hydrogen or C₁-C₆ alkylor R_(c) and R_(d), taken together with the nitrogen atom to which theyare linked, form a morpholino or piperidino ring; or two of R₄, R₅ andR₆ taken together form a C₄-C₁₂ polymethylene chain, which can beunsubstituted or substituted by a C₁-C₁₂ alkyl, by a C₂-C₁₂ alkenyl orby a C₁-C₁₂ alkylidene group, wherein the alkyl, alkenyl and alkylidenegroups may be in turn unsubstituted or substituted by a substituentchosen from halogen, C₁-C₆ alkoxy, hydroxy, cyano, carboxy, (C₁-C₆alkoxy)carbonyl, aryloxy and aryl; the remaining one being hydrogen orC₁-C₁₂ alkyl; or a pharmaceutically acceptable salt thereof; in amountseffective to produce a superadditive immunosuppressant effect, as acombined preparation for simultaneous, separate or sequential use inimmunosuppressant therapy. Said preparation having therefore apotentiated immunosuppressive activity with respect to productscontaining either the immunosuppressive agent (A) or the2,2′-bi-1H-pyrrole immunosuppressive compound (B).

Also disclosed is a combination preparation containing: (a) animmunosuppressant agent (A) and (b) at least one immunosuppressant 2,2-bi-1H-pyrrole compound (B) of formula (I), as defined above, or apharmaceutically acceptable salt thereof, in a quantity producing asuperadditive immunosuppressive effect.

The present invention also provides a pharmaceutical composition for usein immunosuppressant therapy in mammals, including humans, comprising:

(a) an immunosuppressant agent (A) in a pharmaceutically acceptablecarrier and/or excipient, and

(b) at least one immunosuppressive 2,2′-bi-1H-pyrrole compound (B) offormula (I), as defined above, or a pharmaceutically acceptable saltthereof in a pharmaceutically acceptable carrier and/or excipient, inamounts effective to produce a superadditive immunosuppressant effect,said composition having a potentiated immunosuppression activity withrespect to a composition containing either the immunosuppressive agent(A) or the 2,2′-bi-1H-pyrrole immunosuppressant compound (B).

A further aspect of the present invention is an immunosuppressanttherapy method for use in mammals, including humans, in need thereof,the method comprising administering to said mammal (a) animmunosuppressant agent (A) and (b) at least one immunosuppressant2,2′-bi-1H-pyrrole compound (B) of formula (I), as defined above, or apharmaceutically acceptable salt thereof, in amounts effective toproduce a superadditive immunosuppressive effect.

The invention also provides a method for lowering the side effectscaused by immunosuppressant therapy with an immunosuppressant agent (A)or a 2,2′-bi-1H-pyrrole compound (B) in mammals, including humans, inneed thereof, the method comprising administering to said mammal acombination preparation comprising (a) an immunosuppressant agent (A)and (b) at least one 2,2′-bi-1H-pyrrole immunosuppressive compound (B)of formula (I), as defined above, or a pharmaceutically acceptable saltthereof, in a quantity effective to produce a superadditiveimmunosuppressive effect. Accordingly, said combination preparation canbe used for lowering the side effects caused by immunotherapy inmammals, including humans.

In the combined preparations, pharmaceutical compositions and method oftreatment according to the present invention only one immunosuppressant2,2′-bi-1H-pyrrole compound (B), or a pharmaceutically acceptable salttherapy, is preferably used. The combination preparation according tothe invention can also include combination packs or compositions inwhich the constituents are placed side by side and can therefore beadministered simultaneously, separately or sequentially to one and thesame mammal, including humans.

The immunosuppressant agent (A), which is administered with a2,2′-bi-1H-pyrrole compound (B), may be for instance one of thefollowing:

(a) cyclosporin A or cyclosporin C, a non-polar cyclic oligopeptide;

(b) FK506, a fungal macrolide immunosuppressant;

(c) azathioprine, or 6 [(1-Methyl-4-nitro-1H-imidazol-5-yl)thio]1H-purine;

(d) methotrexate;

(e) rapamycin, a fungal macrolide immunosuppressant;

(f) mycophenolate mofetil, or6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methyl-4-(E)-hexenoicacid 2-(4-morpholinyl)-ethyl ester; and

(g) an immunosuppressant glucocorticoid, such as prednisone ordexamethasone;

or a mixture of two or more thereof.

Preferably immunosuppressant agent (A) contains at least one of thefollowing: cyclosporin A, azathioprine, prednisone, dexamethasone ormycophenolate mofetil.

More preferably immunosuppressant agent (A) is cyclosporin A.2,2′-bi-1H-pyrrole compounds (B) of formula (I) as defined above areknown from WO 95/17381, J6 1280429 and J0 2250825 and can be obtained asdescribed therein.

The compounds of formula (I) can be represented also by the followingtautomeric formula (Ia)

wherein R₁, R₂, R₃, R₄, R₅ and R₆ are as defined above; as described inWO 95/17381.

In a compound of formula (I) the substituents have preferably thefollowing meanings. A halogen atom is preferably chlorine or fluorine.The alkyl, alkoxy, alkenyl, alkanoyl, alkenoyl, alkadienoyl andalkylidene groups may be branched or straight chain groups.

An aryl group as a substituent as well as a moiety in an aryloxy,aralkyl or arylcarbamoyl group is, e.g., an aromatic C₆-C₂₀ mono- orpoly-nuclear moiety, typically phenyl, unsubstituted or substituted byone or two substituents independently chosen from halogen, hydroxy,C₁-C₆ alkyl and C₁-C₆ alkoxy.

Accordingly an aralkyl group is e.g. benzyl or phenethyl, in which thephenyl ring is optionally substituted by one or two substituentsindependently chosen from halogen, hydroxy, C₁-C₆ alkyl and C₁-C₆alkoxy.

A C₄-C₁₂ polymethylene chain is e.g. a C₄-C₉ polymethylene chain.

A C₃-C₄ or C₃-C₆ alkenyl group is preferably an allyl group.

A C₁-C₆ alkyl group is preferably a C₁-C₄ alkyl group, in particular amethyl or ethyl group.

A C₁-C₁₂ alkyl group is preferably a C₁-C₆ alkyl group.

An unsubstituted C₁-C₁₁ alkoxy group is preferably a C₁-C₄ alkoxy orC₈-C₁₁ alkoxy group, typically methoxy, ethoxy, propoxy, butoxy andundecyloxy.

A C₁-C₆ alkoxy group substituted by phenyl is preferably a phenyl-C₁-C₄alkoxy group, typically benzyloxy or phenylethoxy.

A C₁-C₂₀ alkyl group is preferably a C₅-C₁₄ alkyl group, in particularan undecyl group.

A C₂-C₂₀ alkenyl group is preferably a C₅-C₁₄ alkenyl group, inparticular an undecenyl group.

A C₂-C₂₀ alkanoyl group is preferably a C₅-C₁₄ alkanoyl group, inparticular an undecanoyl group.

A C₃-C₂₀ alkenoyl group is preferably a C₅-C₁₄ alkenoyl group, inparticular an undecenoyl group.

A C₁-C₁₂ alkylidene group is preferably a C₁-C₈ alkylidene group, inparticular a C₄-C₆ alkylidene group.

A C₂-C₁₂ alkenyl group is preferably a C₃-C₆ alkenyl group.

A (C₁-C₆ alkoxy)carbonyl group is preferably a (C₁-C₄ alkoxy)carbonylgroup. Examples of pharmaceutically acceptable salts of a compound offormula (I) are either those with inorganic bases, such as sodium,potassium, calcium and aluminium hydroxides, or with organic bases, suchas lysine, arginine, N-methyl-glucamine, triethylamine, triethanolamine,dibenzylamine, methylbenzylamine, di-(2-ethyl-hexyl)-amine, piperidine,N-ethylpiperidine, N,N-diethylaminoethylamine, N-ethylmorpholine,β-phenethylamine, N-benzyl-β-phenethylamine, N-benzyl-N,N-dimethylamineand the other acceptable organic amines, as well as the salts withinorganic, e.g. hydrochloric, hydrobromic and sulphuric acids and withorganic acids, e.g. citric, tartaric, maleic, malic, fumaric,methanesulphonic and ethanesulphonic acids.

Preferred 2,2′-bi-1H-pyrrole compounds (B) are the compounds of formula(I), wherein

R₁ is hydrogen or C₁-C₂₀ alkyl;

R₂ and R₅ are hydrogen;

R₃ represents hydroxy or C₁-C₁₁ alkoxy unsubstituted or substituted byphenyl;

R₄ represents hydrogen or C₁-C₄ alkyl;

R₆ is hydrogen, C₁-C₁₄ alkyl or C₂-C₁₄ alkenyl, wherein the alkyl andalkenyl groups may be unsubstituted or substituted by a substituentchosen from halogen, C₁-C₄ alkoxy, hydroxy, phenyl, phenoxy and cyano;

or R₅ and R₆, taken together, form a C₄-C₁₂ polymethylene chain, whichcan be unsubstituted or substituted by a C₁-C₆ alkyl, by a C₃-C₆ alkenylor by a C₁-C₈ alkylidene group, wherein the alkyl, alkenyl andalkylidene groups may be in turn unsubstituted or substituted by asubstituent chosen from halogen, C₁-C₄ alkoxy, hydroxy, cyano, phenoxyand phenyl; and the pharmaceutically acceptable salts thereof.

Specific examples of compounds of formula (I) are the following:

4-methoxy-5-{[5-(undec-10-en-1-yl)-2H-pyrrol-2-ylidene]methyl}-2,2′-bi-1H-pyrrole;

4-ethoxy-5-[(5-decyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-ethoxy-5-[(5-dodecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-ethoxy-5-[(3,5-nonamethylene-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-ethoxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;m.p. 94-96° C.*;

4-propoxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)-methyl]-2,2′-bi-1H-pyrrole;m.p. 73-77° C.*;

4-butoxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;m.p. 81-83° C.*;

4-ethoxy-5-[(5-methyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;m.p. 200° (dec)*;

4-methoxy-5-[(5-decyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;m.p. 100-116° C.*;

4-methoxy-5-[(5-pentadecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;m.p. 100-104° C.*;

4-methoxy-5-[(5-heptyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;m.p. 140-145° C.*;

4-methoxy-5-[(5-phenethyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;m.p. 170° C. dec*;

4-methoxy-5-{[5- (5-carboxy-pent-1-yl) -2H-pyrrol-2-ylidene]methyl}-2,2′-bi-1H-pyrrole; m.p. 157-165° C.*;

4-methoxy-5-{[5- (5-carboxy-pent-1-yl)-2H-pyrrol-2-ylidene]methyl}-2,2′-bi-1H-pyrrole methylester; m.p.138-140° C.*;

4-methoxy-5-[4,5,6,7-tetrahydro-2H-indol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;m.p. 212° C.*;

4-methoxy-5-[(4-hexyl-4,5,6,7-tetrahydro-2H-indol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;m.p. 181-184° C.*;

4-ethoxy-5-{[5-(undec-10-en-1-yl)-2H-pyrrol-2-ylidene]methyl}-2,2′-bi-1H-pyrrole;m.p. 80-97° C.*;

4-methoxy-5-[(4-ethyl-3,5-dimethyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-methoxy-5-[(4-hexyl-5-methyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-methoxy-5-[(5-methyl-4-undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-methoxy-5-[(5-nonyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-methoxy-5-[(5-methyl-4-pentyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-isopropoxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-amyloxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-undecyloxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-benzyloxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;m.p. 90-93° C.*;

4-benzyloxy-5-[(2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole; m.p.200-202° C.*;

4-undecyloxy-5-[(2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-methoxy-5-[(5-tridecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;m.p. 80-100° C.*;

4-ethoxy-5-[(5-tridecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;m.p. 88-93° C.*;

4-buthoxy-5-[(5-tridecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-benzyloxy-5-[(5-tridecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-methoxy-5-[[5-(5-phenoxy-pent-1-yl)-2H-pyrrol-2-ylidene]methyl]-2,2′-bi-1H-pyrrole;m.p. 126-129° C.*;

4-ethoxy-5-[[5-(5-phenoxy-pent-1-yl)-2H-pyrrol-2-ylidene]methyl]-2,2′-bi-1H-pyrrole;m.p. 110-120° C.*;

4-buthoxy-5-[[5-(5-phenoxy-pent-1-yl)-2H-pyrrol-2-ylidene]methyl]-2,2′-bi-1H-pyrrole;

4-benzyloxy-5-[[5-(5-phenoxy-pent-1-yl)-2H-pyrrol-2-ylidene]methyl]-2,2′-bi-1H-pyrrole;

4-methoxy-5-[[5-(6-fluoro-hex-1-yl)-2H-pyrrol-2-ylidene]methyl]-2,2′-bi-1H-pyrrole;m.p. 115-124° C.*;

4-methoxy-5-[[5-(6-hydoxy-hex-1-yl)-2H-pyrrol-2-ylidene]methyl]-2,2′-bi-1H-pyrrole;m.p. 118-121° C.*;

4-methoxy-5-[[5-(5-morpholinecarboxamido-pent-1-yl)-2H-pyrrol-2-ylidene]methyl]-2,2′-bi-1H-pyrrole

NMR (CDCl₃) δppm: 1.2-1.8 (m, 6H); 2.2 (m, 2H); 2.8 (m, 2H); 3.4-3.5 (m,8H); 4 (s, 3H); 6.2 (m, 2H); 6.8 (m, 1H), 7.1 (s, 1H); 7.4-7.6 (m, 3H);12.2-12.4 (bs, 1H); 12.5-12.8 (two bs, 2H); * and

4-methoxy-5-[[5-(7-cyano-hept-1-yl)-2H-pyrrol-2-ylidene]methyl]-2,2′-bi-1H-pyrrole

NMR (CDCl₃) δppm: 1.3-1.8 (m, 10H); 2.3 (m, 2H); 3 (m, 2H); 4.04 (s,3H); 6.1 (d, 1H); 6.2 (dd, 1H), 6.4 (m, 1H); 6.8 (m, 1H); 6.9 (m, 1H);7.03 (s, 1H); 7.25 (m, 1H); 12.6-12.7 (two bs, 2H; 12.9 (bs, 1H); *; andthe pharmaceutically acceptable salts thereof.

The symbol “*” means determined as hydrochloride.

More preferred 2,2′-bi-1H-pyrrole compounds (B) are the following:

4-ethoxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-methoxy-5-[(5-tridecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-ethoxy-5-[(5-tridecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;

4-buthoxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;and

4-benzyloxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;and the pharmaceutically acceptable salts thereof.

As stated above, co-administration of an immunosuppressant agent (A) andof at least one immunosuppressant 2,2′-bi-1H-pyrrole compound (B),produces a potentiated immunosuppressive activity in synergistic way,thus giving a superadditive immunosuppressive effect, i.e. effect whichis grater than the sum of the actions of the individual components.

The superadditive actions of the combination preparations of the presentinvention are shown for instance by the following tests.

M. Tuberculosis Induced Adjuvant Arthritis in Rats

Adjuvant arthritis is induced in groups of 8 male Lewis rats, weighing200 g by injecting 100 μg of M. tuberculosis (H37Rv - heat killed) in 50μl of mineral oil into the plantar surface of the right hind foot pad.The compound4-benzyloxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrolehydrochloride (PNU 156804) is administered at 0.8-0.4-0.2 mg/kg i.v.every other day for a total of 14 administrations, starting on the sameday of the mycobacterium injection. CsA is administered at 5 and 1 mg/kgos every day for 28 days, starting on the same day of the mycobacteriuminjection. When the two compounds are administered in association, thesame schedule of administration is used, the doses tested being 0.4 and0.2 mg/kg i.v. for PNU 156804 and 1 mg/kg os for CsA.

The volumes of the controlateral hind foot pads (systemic,immunologically mediated, disease) are measured pletismographically ondays 0 and 28: the differences represent the oedema volumes. Theactivities of the test compounds are expressed as their capability toinhibit the oedema formation.

The following table summarizes the data obtained in the test.

Oedema Dose volume % Compounds (mg/kg) Route (mm³⁾ inhibition PNU 1568040.8 i.v.  485 66 0.4 i.v. 1512  0 0.2 i.v. 1662  0 CsA 5   os  87 94 1  os 1325  6 PNU 156804 + 0.4 + 1 i.v. + os  175 88 CsA 0.2 + 1 i.v. + os 787 44 Vehicle — i.v. + os 1408 —

These data clearly demonstrate that co-administration of doses by itselfinactive of an immunosuppressant agent (A) i.e. cyclosporin A and of arepresentative immunosuppressant 2,2′-bi-1H-pyrrole compound (B), i.e.PNU 156804, produces a synergic immunosuppressive effect.

Accordingly, the combined preparation of the present invention is aneffective new tool in immunosuppressant therapy. In fact it allowsadministration of lower dosage levels of immunosuppressive agents, thuslowering the side effects caused by commonly used immunosuppressantagents.

The combination preparation of the invention can therefore be used inmammals, including humans, as immunosuppressive agents for theprevention and treatment of rejection phenomena associated with tissueand organ transplantations, graft-versus-host diseases and autoimmunediseases.

Preferred cases of organ and tissue transplants which can besuccessfully treated by the combination preparation of the invention,hereabove described, are, for example, the cases of heart, kidney andbone marrow transplantation.

Preferred cases of autoimmune diseases which can be successfully treatedby the combination preparation of the invention, hereabove described,are for example, the cases of rheumatoid arthritis, systemic lupuserythematosus, juvenile diabetes, autoimmune haemolytic anaemia,miastenia gravis, multiple sclerosis, psoriasis, ulcerative colitis,idiopathic thrombocytopenic purpura, active chronic hepatitis,glomerulonephritis, idiopathic leucopenia, primary biliary cirrhosis,thyroiditis, thyrotoxicosis, dermatomyositis, discoid lupuserythematosus, psoriatic arthritis, regional enteritis, nephroticsyndrome, lupus nephritis, lupoid hepatitis, Sjögren's syndrome,Goodpasture's syndrome, Wegener's granulomatosis, scleroderma, Sezary'sdisease, uveitis and mumps orchitis. Typically rheumatoid arthritis,systemic lupus erythematosus, juvenile diabetes, miastenia gravis,multiple sclerosis and psoriasis.

Given that both component (A) and component (B) of the combinationpreparation according to the present invention have immunosuppressantactivity, the proportions of immunosuppressant agent (A) and ofimmunosuppressant 2,2′-bi-1H-pyrrole compound (B) can be in the range of1:50 to 50:1.

Therefore the dosage of component (A) can vary depending on theconcentration of component (B), and vice-versa. However, otherwisesubactive doses of either immunosuppressant (A) or (B) or of both arepreferably used.

In particular, thanks to the superadditive immunosuppressive effect, theamount of each of agent (A) and compound (B) that is administered ispreferably from about 5 to about 85% of the single amount of eachcomponent that would be administered when given in the absence of theother component, i.e. of its therapeutically effective amount when givenalone, although lower levels of component (A) or component (B) may beadministered.

For instance, when component (A) of the combination preparationaccording to the invention is cyclosporin A, suitable therapy comprises,e.g., i.v. administration of approximately (a) 0.1 to 5 mg/kg,preferably about 0.2 to about 2.5 mg/kg of cyclosporin A and (b)approximately 0.03 to 1.5 mg/kg, preferably about 0.06 mg/kg to about0.7 mg/kg of the immunosuppressant 2,2′-bi-1H-pyrrole compound (B),e.g., PNU 156804. The dose for oral administration in adult humans is ingeneral at most 1 to 15 mg/kg/day of cyclosporin (a) (component (A)),where a serum level of 100 to 200 ng/ml should not be exceeded, and of0.3 to 15 mg/kg/day of the 2,2′-bi-1H-pyrrole compound (component B),e.g. PNU 156804.

The dosage to be used is, of course, dependent on various factors suchas the organism to be treated (e.g., human or animal, age, weight,general state of health), the severity of the symptoms, the disorder tothe accompanying treatment with other pharmaceuticals, or the frequencyof the treatment. The dosages are in general administered several timesper day and preferably once to three times per day. The amounts of theindividual active compounds should be within the range given above, e.g.within the tolerable, efficacious dosage range for the organism to betreated.

The oral route is employed, in general, for all conditions requiring thecompounds of the invention. Preference is given to intravenous injectionor infusion for the acute treatments. For maintenance regimens the oralor parenteral, e.g. intramuscular or subcutaneous, route is preferred.

The nature of the pharmaceutical preparations and compositions accordingto the invention, in which components (A) and (B) can be in the same ordifferent pharmaceutical dosage forms, will of course depend upon thedesired route of administration and physical and chemical compatibilitybetween the two components.

Compounds, i.e. components, (A) and (B) are herein defined as “theactive agents” of the invention.

The compositions may be formulated in the conventional manner with theusual ingredients. For example, the active agents of the invention, maybe administered in the form of aqueous or oily solutions or suspensions,tablets, pills, gelatine capsules, syrups, drops or suppositories.

Thus, for oral administration, the pharmaceutical compositions,containing the active agents of this invention, are preferably tablets,pills or gelatine capsules which contain the active substance togetherwith diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol,cellulose; lubricants, for instance silica, talc, stearic acid,magnesium or calcium stearate, and/or polyethylene glycols; or they mayalso contain binders, such as starches, gelatine, methylcellulose,carboxymethylcellulose, gum-arabic, tragacanth, polyvinylpyrrolidone;disaggregating agents, such as starches, alginic acid, alginates, sodiumstarch glycolate; effervescing mixture; dyestuffs; sweeteners; wettingagents, such as lecithin, polysorbates, lauryl-sulphates and in general,non-toxic and pharmacologically inactive substances used inpharmaceutical formulations.

Said pharmaceutical preparations may be manufactured in known manner,for example by means of mixing, granulating, tabletting, sugar-coating,or film-coating processes.

The liquid dispersions for oral administration may be e.g. syrups,emulsions and suspensions.

The syrups may contain as carrier, for example, saccharose or saccharosewith glycerine and/or mannitol and/or sorbitol.

The suspensions and the emulsions may contain as carrier, for example, anatural gum, agar, sodium alginate, pectin, methylcellulose,carboxymethylcellulose, or polyvinyl alcohol.

The suspensions or solutions for intramuscular injections may containtogether with the active agent a pharmaceutically acceptable carrier,e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propyleneglycol, and if desired, a suitable amount of lidocaine hydrochloride.

The solutions for intravenous injections or infusions may contain ascarrier, for example, sterile water or preferably they may be in theform of sterile aqueous isotonic solutions.

The suppositories may contain together with the active agent apharmaceutically acceptable carrier, e.g. cocoa butter, polyethyleneglycol, a polyoxyethylene sorbitan fatty acid ester surfactant orlecithin.

The following examples illustrate but do not limit the presentinvention.

FORMULATION EXAMPLE 1

Injectable Solution

Component (A): Cyclosporin (A) 75 mg 94% Ethanol and Cremophor EL ® 3 ml

to be diluted with saline or 5% dextrose solution before administration.

Component (B): PNU 156804 25 mg 94% Ethanol and Cremophor EL ® 2 ml

to be diluted with saline or 5 dextrose solution before administration.

The above components (A) and (B) can be placed in separate vials. Thevials can be combined for preparing a solution on actual use.

FORMULATION EXAMPLE 2

Capsules, each dosed at 0.5 g and containing 50 mg of the activesubstance can be prepared.

Composition for 200 capsules:

4-benzyloxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]- 10 g2,2′-bi-1H-pyrrole hydrochloride (PNU 156804) Lactose 80 g Corn starch 5g Magnesium stearate 5 g

This formulation is encapsulated in two-piece hard gelatin capsules anddosed at 0.5 g for each capsule.

FORMULATION EXAMPLE 3

Cyclosporin A: 100 mg

Soft gelatin capsules containing Cyclosporin A 100 mgdispersed/dissolved in a suitable excipient/carrier can be manufacturedaccording to the common galenic technique.

What is claimed is:
 1. A product comprising: (A) cyclosporin A and atleast one immunosuppressant 2,2′-bi-1H-pyrrole compound (B) selectedfrom the group consisting of:4-benzyloxy-5-[(undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;4-benzyloxy-5-[(2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;4-benzyloxy-5-[(5-tridecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrole;and4-benzyloxy-5-[[5-(5-phenoxy-pent-1-yl)-2H-pyrrol-2-ylidene]methyl]-2,2′-bi-1H-pyrrole.2. The product according to claim 1, wherein the amount of each of (A)cyclosporin A and compound (B) is from 5 to 85% of its therapeuticallyeffective amount when given alone.
 3. A method of immunosuppressanttherapy in mammals, in need thereof, comprising administering to saidmammal (A) cyclosporin A and at least one immunosuppressant2,2′-bi-1H-pyrrole compound (B) as defined in claim 1, or apharmaceutically acceptable salt thereof, in an amount effective toproduce a superadditive immunosuppressive effect.
 4. The method of claim3, wherein said mammals are humans.
 5. A method for lowering the sideeffects caused by immunosuppressant therapy in mammals, in need thereof,comprising administering to said mammal a combination preparationcomprising (A) cyclosporin A and at least one 2,2′-bi-1H-pyrrolimmunosuppresive compound (B) as defined in claim 1, or apharmaceutically acceptable salt thereof, in a quantity effective toproduce a superadditive immunosuppresive effect.
 6. The method of claim5, wherein said mammals are humans.
 7. The product according to claim 1,wherein the immunosuppressant compound (B) is4-benzyloxy-5-[(undecyl-2H-pyrrol-2-ylidene)methyl]-2,2′-bi-1H-pyrrolehydrochloride.